LOCUS       sequence-194731-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon system construct with 5 concatenated U6 promoter
            driven transcriptional cassettes for the activation of OCT4. 
            Contains PGK-puro selection cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  AUTHORS   Weltner J, Balboa D, Katayama S, Bespalov M, Krjutskov K, Jouhilahti
            EM, Trokovic R, Kere J, Otonkoski T
  TITLE     Human pluripotent reprogramming with CRISPR activators.
  JOURNAL   Nat Commun. 2018 Jul 6;9(1):2643. doi: 10.1038/s41467-018-05067-x.
  PUBMED    29980666
REFERENCE   2  (bases 1 to 7302)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-07-6"; volume: "9(1):2643. doi"; pages: " 
            10.1038/s41467-018-05067-x"
COMMENT     Sequence Label: PB-GG-OCT4-1-5-PGK-Puro
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    118..151
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        212..709
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(247..270)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     642..661
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             749..1348
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(749..768)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1245..1265
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1429..1446)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1435..1659
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1706..1722)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1730..1746
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1754..1760,1761..1778,1779..1784))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1799..1820
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(1879..2003)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   2293..2327
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2356..2375)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2357..2375)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2385..2402)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2385..2401)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2394..2416)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2543..2998
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2630..2649)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2840..2861
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3024..3128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3129..3197,3198..3989)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3347..3366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4160..4748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4649..4668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5036..5057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5072..5077,5078..5095,5096..5102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5110..5126
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5134..5150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5169..5189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5171..5189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   5240..5302
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        5515..5763
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        5784..5859
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5870..6118
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        6140..6215
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6226..6474
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        6496..6571
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6582..6830
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        6852..6927
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6938..7186
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        7208..7283
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
       61 ccgggcgacc aaaggtcgcc cgacgcccaa ttccgatcat attcaataac ccttaatata
      121 acttcgtata atgtatgcta tacgaagtta ttaggtctga agaggagttt acgtccagcc
      181 aagcttagga tctcgacctc gaaattctac cgggtagggg aggcgctttt cccaaggcag
      241 tctggagcat gcgctttagc agccccgctg gcacttggcg ctacacaagt ggcctctggc
      301 ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct
      361 tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt
      421 cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca
      481 ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct
      541 tcgctttctg ggctcagcag ctgggaaggg tgggtccggg ggcgggctca ggggcgggct
      601 caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa
      661 gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga cctgcatcca
      721 tctagatctc gagcagctga agcttaccat gaccgagtac aagcccacgg tgcgcctcgc
      781 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
      841 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
      901 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
      961 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     1021 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     1081 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     1141 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     1201 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     1261 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     1321 gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga
     1381 aaggagcgca cgaccccatg catcgatgat ctagagctcg ctgatcagcc tcgactgtgc
     1441 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1501 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1561 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     1621 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacct
     1681 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1741 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1801 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1861 tgtcgtgcca gcggatccca tagtgactgg atatgttgtg ttttacagta ttatgtagtc
     1921 tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt
     1981 tcagcttttt tgtacaaact tgtgctagca ttctagttga tatctataac aagaaaatat
     2041 atatataata agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt
     2101 taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat
     2161 agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg
     2221 cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat
     2281 atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta atctagaatg
     2341 gcgcgcccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     2401 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     2461 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2521 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2581 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2641 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2701 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2761 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2821 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2881 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2941 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     3001 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3061 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     5101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     5161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggattaatta agcggccgca
     5221 tacgcgtata ctagattaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa
     5281 tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat
     5341 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa
     5401 caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata
     5461 aagtaacaaa acttttaact agaggatctc gagctcaagc ttcgaattcc ccctgagggc
     5521 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     5581 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     5641 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     5701 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     5761 accgggggga gaaactgagg cgagttttag agctagaaat agcaagttaa aataaggcta
     5821 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttcatgg agggcctatt
     5881 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     5941 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     6001 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     6061 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg
     6121 gtggtggcaa tggtgtctgg ttttagagct agaaatagca agttaaaata aggctagtcc
     6181 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tggacgaggg cctatttccc
     6241 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     6301 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     6361 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     6421 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggacac
     6481 aactggcgcc cctccgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     6541 tcaacttgaa aaagtggcac cgagtcggtg cttttttcca ggagggccta tttcccatga
     6601 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     6661 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     6721 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     6781 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gggcacagtg
     6841 ccagaggtct ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     6901 cttgaaaaag tggcaccgag tcggtgcttt ttttgttgag ggcctatttc ccatgattcc
     6961 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     7021 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     7081 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     7141 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgtct gtgggggacc
     7201 tgcactggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     7261 aaaaagtggc accgagtcgg tgctttttta acgagcggcc gc
//
